1. Field of the Invention
The present invention relates generally to solid substrates for conducting biological assays and more specifically to assay beads (microbeads) and methods for use thereof to carry out multiplexed bioassays. The invention particularly relates to multiplexed bioassays using micro-volume samples, such as protein and nucleic acid analysis. The invention further relates to photo-curable epoxy compositions such as those containing EPON SU-8 epoxy resin (Hexion Specialty Chemicals), EPON 1002F epoxy resin (Hexion Specialty Chemicals), as well as other bi-functional or multifunctional epoxy resins. Preferred photo-curable compositions exhibiting significantly better performance according to the invention further include carboxyl-containing monomers such as acrylic acid, 2-carboxyethyl acrylic acid, 4-vinylbenzoic acid, or 3-acrylamido-3-methyl-1-butanoic acid, or glycidyl methacrylate, and the like. The photo-curable compositions may be used to cast films or fabricate beads, magnetic beads, or magnetic beads containing nickel barcodes. The resulting various kinds of films, micro beads, magnetic beads, or magnetic beads containing nickel barcodes and are useful in a variety clinical or biological applications.
2. Description of Related Art
As current research in genomics and proteomics produces more multiplexed data, there is a need for technologies that can rapidly screen a large number of nucleic acids and proteins in a very small volume of samples. Microarray, DNA chips, and protein chips have drawn a great deal of commercial interest. The assays are typically performed on a planar biochip platform by arraying and immobilizing DNA on a solid support via mechanical printing in the x-y position onto the microscope slide, by piezoelectric ink-jetting or by direct synthesis of DNA on the chip. However, mechanical contact printing is not very desirable because it prints one spot per contact that results in relatively large printing variations from spot to spot or batch to batch, inconsistent spot morphology, misprinting, and slide surface variations, all of which are undesirable for DNA microarray analysis. Further, distributing a small volume of liquid samples over a relatively large chip surface often encounters the problems of insufficient sample amounts or non-uniform distribution over the chip surface. These problems can cause incomplete reactions or very long reaction time.
Micro bead technology potentially overcomes many of the problems of microarray technology and provides better quality control of each probe, flexibility with the assembly of various type and amount of probes in an analysis, and convenience of doing analysis without mechanical printing. Existing micro bead approaches include (1) the incorporation of spherical beads or particles with spectrally distinguishable fluorophore, fluorescent semiconductor quantum dots, and (2) metallic rods with either bar coded color (absorption) stripes or black and white strips. Both fluorescence and barcode strip beads are identified by optical detection in reflective or emissive configuration. The problems of reflection configuration are (1) it is difficult to setup the collection optics in proper position, especially when the bead dimensions are on a micrometer scale, and (2) the light collection efficiency is poor and the barcode contrast is low, especially when micro beads are in the micro flow system. The flow scatters light, which interferes with optical reflection or emissive detection. Further, fluorescent beads, the spectral range and the possible number of spectrally distinguishable labels, however, often limit the potential number of code variations. Many laser light sources are often needed to excite different fluorescent labels. In addition, the validity of the coding signatures is another serious concern, since the incorporated coding elements in some cases may be lost; photo bleached, or interfered spectrally with the analytical signals. In the case of multi-metal (Au, Pt, Ni, Ag, etc) color micro rods, the encoding scheme suffers from the difficulty of manufacturing and the number of colors, based on different metal materials, is limited. Many 1D or 2D bar codes are recognized by their specific image patterns. Optical imaging method is used for recognition of these bar code patterns. Although high speed camera is available for capturing bar code images, pattern recognition is a slow and time consuming process. It often needs special software to analyze the images section by section. Therefore, it is difficult to identify hundreds or thousands of beads in a short time to improve throughput. The following patent documents disclose some of the systems that exhibit some of the deficiencies noted above.
U.S. Pat. No. 6,773,886 issued on Aug. 10, 2004, the entire contents of which are incorporated herein by reference, discloses a form of bar coding comprising 30-300 nm diameter by 400-4000 nm multilayer multi metal rods. These rods are constructed by electrodeposition into an alumina mold; thereafter the alumina is removed leaving these small multilayer objects behind. The system can have up to 12 zones encoded, in up to 7 different metals, where the metals have different reflectivity and thus appear lighter or darker in an optical microscope depending on the metal type whereas assay readout is by fluorescence from the target, and the identity of the probe is from the light dark pattern of the barcodes.
U.S. Pat. No. 6,630,307 issued on Oct. 7, 2003, the entire contents of which are incorporated herein by reference, discloses semiconductor nano-crystals acting as a barcode, wherein each semiconductor nanocrystal produces a distinct emissions spectrum. These characteristic emissions can be observed as colors, if in the visible region of the spectrum, or may be decoded to provide information about the particular wavelength at which the discrete transition is observed.
U.S. Pat. No. 6,734,420 issued on May 11, 2004, the entire contents of which are incorporated herein by reference, discloses an identification system comprising a plurality of identifiable elements associated with labels, the labels including markers for generating wavelength/intensity spectra in response to excitation energy, and an analyzer for identifying the elements from the wavelength/intensity spectra of the associated labels.
U.S. Pat. No. 6,350,620 issued on Feb. 26, 2002, discloses a method of producing a micro carrier employing the shape, size, and color of the carrier as image bar codes for identification. The patent discloses an identification system comprising a bar code is formed on the substrate by photolithography, and then using nickel plates to hot compress the bar code onto the surface of bead to form a microcake-like particle. The bar code pattern can be classified by an imaging recognition system.
U.S. Pub. No. US2005/0003556 A1, the entire contents of which are incorporated herein by reference, discloses an identification system using optical graphics, for example, bar codes or dot matrix bar codes and color signals based on color information signal for producing the affinity reaction probe beads. The color pattern is decoded in optical reflection mode.
U.S. Pub. No. US2005/0244955, the entire contents of which are incorporated herein by reference, discloses a micro-pallet which includes a small flat surface designed for single adherent cells to plate, a cell plating region designed to protect the cells, and shaping designed to enable or improve flow-through operation. The micro-pallet is preferably patterned in a readily identifiable manner and sized to accommodate a single cell to which it is comparable in size.
What is needed is a digitally encoded micro bead that provides for high contrast and high signal-to-noise detection, and that provides for parallel and high-throughput bioanalysis, e.g., of proteins, pathogens, gene expression, single nucleotide polymorphism, nucleic acid-based tissue typing, cell or chromosome sorting, and transcriptional profiling that requires smaller volumes of fluid and rapid assay.
The barcode microbeads or micro pellets are typically fabricated by photo-lithography. Thousands or millions of micro beads or micro patterns can be synthesized on a micro slide, a glass or a silicon wafer. Suitable materials for the fabrication of microbeads include photosensitive photopolymer or so called photoresists, such as EPON 1002F or SU-8 brand epoxy resins. The starting materials can be monomer or polymer, and resulting into cross-linking polymer after UV or photon exposure. Although photopolymers are commonly used in the semiconductor industry, many semiconductor industry photopolymers are not biocompatible because of the difficulty of immobilizing biomolecules, such as proteins, oligonucleotides or cells, on the surface of these materials, especially if long term stability is required for storage. Other problems associated with current photoresists include high auto fluorescence, brittleness, and poor adhesive properties for multilayer formation. More importantly, because the microbeads are suspended in the reaction solution it is desired that all surfaces be bio-reactive. Thus the whole microbead should have the same surface chemical property, unlike a single side surface, such as a film on a glass.
EPON SU-8 epoxy, (Hexion Specialty Chemicals) is a photoresist resin which has been used in microelectromechanincal system (MEMS) for the fabrication of high aspect ratio structure. A solution containing SU-8 resin, photo acid generator, such as triphenyl sulfonium hexafluoroantimonate, and solvent, such as γ-butyrolactone or cyclopentanone is coated onto various substrates, pre-baked to evaporate the solvent, leaving a solid photoresist film of up to several hundred microns thickness depending on the solid content of the solution. By exposing the film through a photo mask to UV irradiation, a pattern is transferred to the photoresist. A high resolution three dimensional negative image of the mask is formed by subsequent immersion into a developer solution. Because the surface of SU-8 has epoxy groups it has hydrophobic properties and presents a limitation to many biological applications requiring specific functional groups. These limitations include challenges in surface wetting, biofouling and limited cell attachment. There remains a need to improve the biocompatibility of such epoxy resins.